Pichia pastoris, a fast growing microbe, though easily cultivable to high cell densities, has a major drawback of relatively lower specific productivity (Porro et al., 2011). The disadvantages associated with the low productivity of the prior art have been remedied in the instant disclosure.
Robustness in the microbial process designed was introduced by using critical nutrient ratio (CNR) of two important nutrients (Tiwari et al., 2012) as a control variable for key metabolic variables like activity of a centrally important regulatory enzyme—phosphofructokinase, oxygen uptake rate as well as specific productivity. As CNR is a general parameter, it is likely to influence synthesis of degrading enzymes and transporter enzymes, just as it affects productivity of the main product.
Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel 34S labeling by Martin Pfeffer et al. characterizes intracellular protein formation, degradation and secretion under a steady state condition. Apart from limitation of chemostat mode of fermentation, their study is also limited to use of limiting substrate methanol instead of using critical nutrient ratio (CNR). Further, their study does not consider the losses due to extracellular degradation and hence ignores a vital aspect of protein dynamics during expression.
Hence, there exists a need in the art to include more relevant parameters in the process design space for manufacturing of recombinant peptides. In the present disclosure we have disclosed use of CNR to regulate synthesis, intracellular and extracellular degradation, secretion of recombinant peptide expressed in fed-batch mode.